Interpretive Summary: Shiga-toxin producing Escherichia coli (STEC) are important foodborne pathogens. E. coli O157:H7, the most frequently isolated STEC serotype, is one of the leading causes of foodborne diseases. Meanwhile, many non-O157 STEC serotypes have been associated with serious outbreaks and clinical infections as well. Various STEC serotypes are capable of forming biofilms on food-contact surfaces that, when detached, may lead to food contamination. In nature, bacteria may coexist in multi-species communities to form mixed biofilms, and the interactions among species could profoundly affect biofilm development, composition and structure. In the present study, we investigated how coexistence of STEC O157:H7 and O26:H11, a high virulent non-O157 STEC serotype, would affect bacterial growth competition and mixed biofilm composition. Our results indicated that STEC O26:H11 strains were able to effectively outgrow O157:H7 strains during coexistence. In the presence of pre-formed O157:H7 biofilms, the O26:H11 strains could still compete with the colonized O157:H7 cells and establish themselves in the mixed biofilms, while STEC O157:H7 appeared to be a poor colonizer if O26:H11 cells pre-occupied the solid surfaces. Our findings highlight the potential risk of this non-O157 STEC in food safety and call for increased attention to the non-O157 STEC biofilms in food processing facilities.